Tumor genotyping assays by NGS were performed using a GENEREAD DNASEQ TARGETED PANELS V2 HUMAN COMPREHENSIVE CANCER PANEL (Qiagen). According to the manufacturer’s instructions, each sample used for sequencing, including cfDNA, contained 40 ng DNA. The PCR products and constructed libraries were analyzed for size range and molarity using an AGILENT 2100 BIOANALYZER. Sequencing was performed using MISEQ with a MISEQ REAGENT KIT V2 (300-CYCLES) for paired-end 151-base pair and HISEQ 2000 with paired-end 100-base pair platforms (Illumina). The data were analyzed using GENEREAD DNASEQ ANALYSIS SOFTWARE (Qiagen) for read mapping and post-processing, variant calling and filtering, and annotation. For variant calling and filtering, the GATK Unified Genotyper program (GATKLite version 2.3-9) was used for calling variants on individual samples, and Strelka46 was used with default parameters to call somatic variants from matched tumor and normal samples. All of the genetic alterations detected in each sample were reported with high, medium or low confidence, according to Qiagen’s GENEREAD DNASEQ ANALYSIS SOFTWARE (Qiagen). The detected gene aberrations were distinguished per nucleotide changes, not only per genes. Further analysis was performed only on high confidence genetic alterations. We identified somatic alterations in tumor tissues to exclude germline alterations, derived from the patient’s germline DNA, observed in crude alterations prepared from each tumor. To compare the genetic alterations between tumor samples, we used an original script in “Terminal app”, a terminal emulator in the MacOS operating system (Supplementary File 32). The terminal command dictated coordination of the same genetic alteration between samples.

Within individual patients, the concordance and discordance of each gene mutation between tDNA and cfDNA was analyzed, and genetic alterations shared among all tumor tissues (primary lesions and all metastatic lesions) and cfDNA samples were selected. Then, the genetic alterations were divided into 3 groups according to the detection frequency in tumor samples from a single patient. Mutations present in all tumor sites including the primary lesion and metastases were defined as truncal. Those present in more than 2 sites but not all sites were defined as shared branch mutations. Those present at only a single site were defined as individual branch mutations. Differences in the concordance between tDNA and cfDNA in the 3 groups were analyzed by Student’s t-test. Correlations between the VAF of a mutation and its detection in cfDNA were analyzed by Student’s t-test. The VAF was calculated based on the allele coverage as follows: VAF = (allele coverage of the variant call)/(sum of the allele coverage of the reference and that of the variant call). We used the Mann–Whitney U test to compare the VAF of a genetic alteration detected in both tDNA and cfDNA with that of an alteration detected in tDNA only (and not cfDNA).

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